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WO1998018940A1 - Expression de genes specifiques aux feuilles dans des plantes transgeniques - Google Patents

Expression de genes specifiques aux feuilles dans des plantes transgeniques Download PDF

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Publication number
WO1998018940A1
WO1998018940A1 PCT/EP1997/005900 EP9705900W WO9818940A1 WO 1998018940 A1 WO1998018940 A1 WO 1998018940A1 EP 9705900 W EP9705900 W EP 9705900W WO 9818940 A1 WO9818940 A1 WO 9818940A1
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Prior art keywords
promoter
plants
plant
leaf
sequence
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PCT/EP1997/005900
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German (de)
English (en)
Inventor
Uwe Sonnewald
Marcus Ebneth
Ralf-Michael Schmidt
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Basf Aktiengesellschaft
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Application filed by Basf Aktiengesellschaft filed Critical Basf Aktiengesellschaft
Priority to AU69091/98A priority Critical patent/AU743558B2/en
Priority to EP97948804A priority patent/EP0938569B1/fr
Priority to US09/284,418 priority patent/US6229067B1/en
Priority to JP10520030A priority patent/JP2001502551A/ja
Priority to BR9712656-0A priority patent/BR9712656A/pt
Priority to CA2268860A priority patent/CA2268860C/fr
Priority to DE59712978T priority patent/DE59712978D1/de
Priority to IL12946497A priority patent/IL129464A/xx
Publication of WO1998018940A1 publication Critical patent/WO1998018940A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/8223—Vegetative tissue-specific promoters
    • C12N15/8225—Leaf-specific, e.g. including petioles, stomata
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters
    • C12N15/8234—Seed-specific, e.g. embryo, endosperm

Definitions

  • the present invention relates to nucleic acid sequences with promoter activity which bring about a leaf-specific expression of coding nucleotide sequences controlled by them in plants, expression cassettes, recombinant vectors and microorganisms which comprise such regulatory sequences, transgenic plants transformed therewith, a method for producing transgenic plants Plants and a method for isolating the leaf-specific promoter.
  • Transgenic plants are currently used in various biotechnological areas. The primary goals are on the one hand plant protection and on the other hand an increase in the quality of the harvestable products.
  • regulation signals are necessary which enable proper transcription. These include promoters and terminators. The terminators located at the 3 'end of the coding DNA serve to terminate the transcription and, if appropriate, as a signal for polyadenylating the mRNA formed. Promoters contain recognition sequences for RNA polymerases and for transcriptional effectors. The promoters are responsible for the expression behavior of the foreign genes.
  • Herbicide-tolerant plants as are known from DE-A-3701623, represent an example of genetic protection measures. Further examples are insect-resistant plants (Vaek et al. (1987) Plant Cell 5, 159-169), virus-resistant
  • inducible expression has been described: wound induction (DE-A-3843628, DE-B-3837752), chemical induction (Ward et al. (1993) Plant Molec. Biol. 22, 361-366) and light induction ( Fluhr et al. (1986) Science 232, 1106-1112).
  • a promoter is known from DE-A-4207358 which effects a lock cell-specific gene expression but no specific expression in mesophyll cells or epidermal cells of leaves.
  • the gas exchange can be regulated as desired by artificially changing the opening periods of the stoma. Herbicide tolerance or resistance cannot be imparted by such a promoter.
  • cell- and tissue-specific expression are: seed-, tuber- and fruit-specific (summarized in Edwards and Coruzzi (1990) Annu. Rev. Genet. 24, 275-303; DE-A-3843627), phloem-specific (Schmülling et al . (1989) Plant Cell 1, 665-670), root lump-specific (DE-A-3702497) and meristem-specific (Ito et al. (1994) Plant Molec. Biol. 24, 863-878) expression.
  • promoters in chloroplast-containing cells are also from Edwards and Coruzzi (1990), Annu. Rev. Genet. 24, 277-279.
  • the promoters described therein cause expression either only in inducible form (e.g. the rbcS-3A promoter) or only in certain cell types (e.g. the GS2 and GS3A promoters), but the expression is not restricted to certain parts of the plant.
  • promoters described are often problematic. Promoters that cause constitutive expression of the genes they control can be used, for example, to produce herbicide-tolerant and pathogen-resistant plants, but have the disadvantage that the products of the genes they control are present in all parts of the plant, including in the harvested parts of the plant, which can be undesirable in some cases. Inducible promoters are also not without problems since the induction conditions in agricultural used plants are typically difficult to control outdoors.
  • the promoter of the phosphoenolpyruvate carboxylase originating from a C4 plant leads to expression in the mesophyll of the leaf (Stockhaus et al., (1994), Mol. Gen. Genet. 245, 286-293).
  • this promoter mediates only low activity in mesophyll. It also shows activity in roots. This low organ specificity is undesirable for many applications.
  • Promoters which cause leaf-specific, preferably permanent expression of genes which are controlled by them are not known.
  • the object of the invention is therefore to provide means which enable targeted organ-specific gene expression in plants.
  • These agents should be suitable, for example, for the expression of resistance genes and genes which modify the photosynthetic performance.
  • This object was surprisingly achieved by providing a new promoter which, in plants, brings about, preferably permanent, leaf-specific expression of a coding nucleotide sequence controlled by it, independently of induction factors.
  • Figure 1 (A) is a schematic representation of the approximately 7100 bp BamHI fragment of the cloned into the vector pUC19
  • FIG. 2 shows the nucleotide sequence of the cy-FBPase promoter from potato. The region which is complementary to the 3 'end of the 5' sub-fragment of the cy-FBPase used for the southern hydride is underlined; two palindromic sequences are underlined; the 5 '-terminal sequence "GGATC" was added to the genomic DNA to create a BamHI interface;
  • Figure 3 (A) shows the construction scheme of the plasmids FBP: GUS and
  • FBP GUS (DEL);
  • B a schematic representation of the plasmid FBP: GUS, with the approx. 1700 bp FBPase promoter, the approx. 1870 bp GUS gene and the approx. 260 bp nopaline synthase terminator, animals in vector pBI 101;
  • FIG. 4 shows a bar diagram which shows the leaf-specific GUS activity in transgenic potato plants which is controlled by the cy-FBPase promoter; the results determined for two different transformation experiments with FBP: GUS (plant line “Me 1-22" and “Me 1-9") are shown and compared with a control experiment ("control");
  • FIG. 5 shows a bar diagram which shows the leaf-specific GUS activity in transformed tobacco plants which is controlled by the cy-FBPase promoter.
  • TME-1/67 denotes the results obtained with the FBP: GUS vector.
  • TME-11/13 denotes the results obtained with the FBP: GUS (DEL) vector.
  • WT shows the results determined for the wild type. Specified is the amount made
  • FIG. 6 shows the histochemical detection of GUS activity in various tissues of the tobacco leaf of a transgenic plant.
  • A Cross-section through the central guiding tissue of the source leaf
  • B Epidermis
  • C Cross-section through the petiole
  • D Cross-section through the mesophyll of a source leaf. The sections were fixed in 3% paraformaldehyde solution for 20 min and then incubated in X-Gluc solution overnight. The chlorophyll was then removed by 70% ethanol;
  • FIG. 7 shows a Northern blot, which shows the uniform leaf-specific GUS expression in transgenic tobacco plants, mediated by the cy-FBPase promoter from potato;
  • FIG. 8 shows histochemical detection of the ⁇ -glucuronidase (GUS) activity in tobacco seedlings;
  • FIG. 9 shows a schematic representation of the plasmid pBin-FBP, with the 1724 bp cy-FBPase promoter from potato and the 280 bp octopine synthase terminator, inserted into vector pBin 19; and FIG. 10 cDNA probe of the cy-FBPase gene from potato (EMBL No.: X76946).
  • the term “gene” or “coding (nucleotide) sequence” denotes a nucleotide sequence which has a specific, optionally inheritable, structure, for example at least one protein, at least one ribozyme or at least one antisense RNA; or function, such as Resistance, coded; or a change in the composition of herbal ingredients, such as oils, fats, enzymes, proteins, biopolymers, so that, for. B. the nutritional value, the yield or the industrial usability of the plant is improved.
  • a “promoter” denotes a nucleotide sequence region which controls the transcription of a gene or the synthesis of the corresponding mRNA.
  • the promoter comprises a sequence 5 'upstream from the transcription start point. As an essential sequence element, it comprises at least the so-called "TATA" box. Other regulatory elements such as the "CAAT” box or a GC box can also be included.
  • the promoter sequence according to the invention in addition to the sequence section mentioned above, is a sequence 3 ′ downstream from the transcription start point, such as, for. B. has a leader sequence or a portion thereof to show the desired promoter activity and / or specificity or to fully develop.
  • Resistance in the context of the present invention means the artificially induced resistance or tolerance of the transgenic plants to herbicides and / or pathogens, such as e.g. Fungi, viruses or insects, against certain external conditions such as high concentrations of ozone, sulfur dioxide, nitrogen oxides or other exogenous pollutants as well as against heat, cold, dryness or UV light.
  • herbicides and / or pathogens such as e.g. Fungi, viruses or insects
  • a "modification of a plant's photosynthetic performance” involves reducing and, in particular, increasing the photosynthetic activity of the transformed plants. This can be done, for example, by expressing genes which specifically increase the light yield of the plant, increase the rate of conversion of individual rate-determining metabolic steps, or influence the exchange of substances with the environment.
  • leaf specificity means that a foreign gene under the control of a promoter according to the invention is expressed in the entire leaf organ or in certain tissues of the leaf, preferably in mesophyll (for example palisade parenchyma), but not in the shoot or in other parts of plants, in particular the roots.
  • mesophyll for example palisade parenchyma
  • “leaf specificity” is also given in the context of the present invention if the promoter according to the invention expresses a foreign gene in the leaf, preferably in the mesophyll of leaves, in particular of source leaves, in comparison with other plant parts, such as stem, non-germinating tubers , Fruits or seeds of the plant favored and in leaves a significant, such.
  • B. causes at least about 5 to 10 times, such as about 10 to 100 times, increased expression.
  • Source leaves of a plant are the older leaves of a plant, which fix excess carbon by photosynthesis and thus bound carbon in other parts of the plant, such as. B. export the younger "Sink” sheets.
  • “permanent” expression means the expression of the gene which is under the control of the promoter according to the invention and is essentially independent of exogenously applied chemical induction signals over one or more plant generations.
  • a first object of the present invention relates to promoters which bring about, preferably permanent, leaf-specific expression of coding nucleotide sequences controlled by them in plants.
  • the primary site of action of herbicides and a large number of pathogens is the leaf tissue, so that a leaf-specific expression of the corresponding resistance genes would offer sufficient protection. Since photosynthesis also takes place in the leaf tissue, the leaf-specific expression of one or more genes which influence the photosynthesis performance would be necessary to modify and in particular to improve the photosynthetic performance.
  • the promoters according to the invention now offer the surprising advantage of being able to express resistance genes specifically at the actual site of action in the plant.
  • preferred promoters allow for the first time the specific localization and expression of a foreign gene in the mesophyll of leaves, in particular of source leaves, while no activity can be observed in parenchymal tissue, as well as xylem, phloem and others.
  • a promoter according to the invention can be provided by isolating and characterizing promoters in leaf-specific and preferably permanently expressed genes.
  • Preferred are promoters which essentially correspond to the promoters of the cytosolic fructose-1,6-bisphosphatase genes (cy-FBPase genes) from leaf-specific mesophyll cells of plants.
  • cy-FBPase genes cytosolic fructose-1,6-bisphosphatase genes
  • cy-FBPase genes cytosolic fructose-1,6-bisphosphatase genes
  • a preferred embodiment relates to a nucleotide sequence with the desired promoter activity, which is isolated from Solanum tuberosum var. Desiree, or functional equivalents thereof.
  • a promoter with a nucleotide sequence selected from SEQ ID NO: 1 to 5 or functional equivalents of these sequences is particularly preferred.
  • the start of transcription in the preferred nucleotide sequence according to SEQ ID NO: 1 was carried out with the aid of "Primer Extension" at A.L.F. (Automatic Laser Fluorescence DNA Sequencer (Pharmacia)).
  • A.L.F. Automatic Laser Fluorescence DNA Sequencer (Pharmacia)
  • a 5 'fluorescent-labeled oligonucleotide was prepared which is complementary to a region of 21 bp in the promoter from +1577 to +1599 (SEQ ID NO: 1).
  • SEQ ID NO: 1 The help of this printer, total RNA from source sheets was rewritten into single-stranded cDNA.
  • the RNA not recognized by the primer and the RNA components of the cDNA / RNA hybrids were then digested.
  • the cDNA was then released on A.L.F.
  • the sequence SEQ ID NO: 1 accordingly comprises 1428 bp promoter region (SEQ ID NO: 4) and 292 bp 5 'untranslated region of the cyt FBPase.
  • a TATA box sequence could be found 30 bp above the starting point ("TTATAAA”), and 141 bp above the CAP box ("ATCATCCAAACAT").
  • TTATAAA starting point
  • ATCATCCAAACAT CATCCAAACAT
  • the position information is based on the numbering of the nucleotide residues according to SEQ ID NO: 2.
  • the above listing contains in particular two 10 bp almost identical palindromic sequence sections, each containing the motif TGCA twice.
  • This motif is in the opposite strand as ACGT, a box that has been used by various working groups as a regulatory sequence (e.g. Guliano et al., (1988), Proc. Acad. Natl. Sei. USA, 85, 7089-7093) and as a binding site for DNA-binding leucine zipper proteins (e.g. Armstrong et al., (1992) Plant Cell, 4, 525-537). The binding of leucine zipper proteins is unlikely to be affected by the orientation of the motif. These sequences are marked by dotted underlining in FIG. 2.
  • Another preferred embodiment of the invention relates to a 5 'truncated promoter sequence (SEQ ID NO: 3). It comprises an 817 bp promoter region (SEQ ID NO: 5) and a 292 bp 5 'untranslated region.
  • SEQ ID NO: 3 a 5 'truncated promoter sequence
  • the shortened promoter surprisingly shows an identical organ or tissue specificity as the longer promoter described above, but has different promoter activity.
  • Functionally equivalent promoter sequences are, according to the invention, those sequences which, despite the different nucleotide sequence, still have the desired functions, i. e. Have promoter activity and tissue or organ specificity.
  • a measure of the promoter activity is, for example, the expression rate determined for a specific marker gene which is under the regulatory control of the promoter according to the invention.
  • suitable marker genes are the ⁇ -glucuronidase (GUS) gene from E. coli or the green fluorescence protein (GFP) gene (Baulcombe et al., (1993), Plant J., 7 (6), 1045 -1053).
  • GUS ⁇ -glucuronidase
  • GFP green fluorescence protein
  • the organ or tissue specificity can easily be determined by comparing the expression rates determined for individual tissues or organs of the plant for the above marker genes.
  • Functional equivalents in the context of the present invention include naturally occurring variants of the sequences described herein as well as artificial, e.g. Artificial nucleotide sequence
  • a functional equivalent is also understood to mean, in particular, natural or artificial mutations in an originally isolated promoter sequence which continue to show the desired function. Mutations include substitutions, additions,
  • the present invention also includes those nucleotide sequences which are obtained by modifying the nucleotide sequence according to SEQ ID NO: 1 to 5.
  • the aim of such a modification can e.g. further narrowing down the promoter sequence contained therein, or e.g. also be the insertion of further restriction enzyme interfaces.
  • Functional equivalents are also those promoter variants whose promoter function is weakened or enhanced compared to the wild type.
  • a genomic bank can be made Sheets of the donor organism can be created by known methods, for example by isolating the total DNA, subsequent partial digestion, packaging fragments of a defined size in bacteriophages, infection of bacteria with the recombinant bacteriophages and subsequent amplification of the genomic library.
  • the phages containing the genomic DNA can then be transferred, for example, to nylon filters and hybridized with a radioactively labeled cDNA of the previously identified leaf-specific gene.
  • Hybridizing phage DNAs can be visualized by autadiadiography and then separated.
  • lytic agar plates can be inoculated and incubated, starting from a single plaque, and the DNA can be obtained in a manner known per se, for example by phenol-chloroform extraction and subsequent precipitation with ethanol.
  • the fragment lengths of the promoter regions of the isolated genomic clones can now be determined, for example, by Southern hybridizations with a 5 'cDNA sample of the leaf-specifically expressed gene after different restriction cleavages.
  • a promoter region can now be cloned into a suitable vector, for example multiplied in E. coli, and the complete nucleotide sequence of the promoter can be determined by sequencing.
  • Suitable cloning vectors include pBR332, pUC series, M13mp series and pACYC184.
  • the promoter can now be operatively linked in an expression cassette to a suitable gene, so that the promoter can control the transcription of the gene fused with it.
  • An operative link is understood to mean the sequential arrangement of promoter, coding sequence, terminator and, if appropriate, further regulatory elements, each of the elements mentioned being able to perform its function as intended for gene expression.
  • Such an expression cassette represents a further subject of the present invention.
  • Another subject of the invention relates to recombinant vectors, e.g. Plasmids or viruses which contain at least one expression cassette according to the invention.
  • the nucleotide sequences which follow the promoter sequence of the expression cassette can contain all possible open reading frames for any peptide and one or more introns.
  • examples include: sequences for enzymes; Sequences that are complementary to a) a genome sequence, the genome sequence being allowed to be an open reading frame; b) an intron; c) a non-coding leader sequence; d) each sequence that - complementarily integrated into the genome - the Transcription, mRNA processing (eg splicing) or the
  • the inserted nucleotide sequence can be produced synthetically or obtained naturally or contain a mixture of synthetic and natural DNA components.
  • synthetic nucleotide sequences are generated with codons that are preferred by plants. These plant preferred codons can be determined from the highest protein frequency codons expressed in most interesting plant species.
  • various DNA fragments can be manipulated in order to obtain a nucleotide sequence which expediently reads in the correct direction and which is equipped with a correct reading frame.
  • adapters or linkers can be attached to the fragments.
  • the promoter and terminator regions according to the invention should expediently be provided in the transcription direction with a linker or polylinker which contains one or more restriction sites for the insertion of this sequence.
  • the linker has 1 to 10, usually 1 to 8, preferably 2 to 6, restriction sites.
  • the linker has a size of less than 100 bp, often less than 60 bp, but at least 5 bp within the regulatory ranges.
  • the promoter according to the invention can be both native or homologous and foreign or heterologous to the host plant.
  • the expression cassette according to the invention contains, in the 5 '-3' transcription direction, the promoter according to the invention, any sequence and a region for the transcriptional termination. Different termination areas are interchangeable.
  • Particularly suitable coding nucleotide sequences are tolerance or resistance-mediating genes, genes which increase the photosynthetic performance of the plant or marker genes such as that ⁇ -glucuronidase gene (GUS) from Escherichia coli.
  • Suitable tolerance genes are, for example, those that increase the temperature, dry or UV tolerance or the tolerance to environmental pollutants of a plant.
  • Suitable resistance genes are, for example, the bar gene from Streptomyces hygroscopicus, which confers resistance to the total herbicide phosphinothricin, chitinase genes, which impart tolerance to fungal infections, and riboyzyme genes, whose RNA transcripts recognize and cleave viral RNA with high specificity can.
  • Suitable genes for increasing the photosynthetic performance are, for example, the genes coding for sucrose phosphate synthase (SPS) or fructose-1,6-bisphosphatase (FBPase).
  • SPS sucrose phosphate synthase
  • FBPase fructose-1,6-bisphosphatase
  • the fused construct can now be transferred into plant genomes by various known methods. Suitable methods are, for example, protoplast transformation by polyethylene glycol-induced DNA uptake, electroporation, sonication or microinjection, and the transformation of intact cells or tissue by micro or macroinjection into tissue or embryos, tissue electroporation, incubation of dry embryos in DNA-containing solution, biolistic gene transfer and particularly preferably Agrobacterium transformation.
  • the methods mentioned are described, for example, in B. Jenes et al. , Techniques for Gene Transfer; in Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S-d Kung and R. Wu, Academic Press, 1993, pp. 128-143 and in Potrykus (1991) Annu. Rev. Plant Physiol.
  • the fused construct is preferably cloned into a vector which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al. (1980) Nucl. Acids Res. 12, 8711).
  • a vector which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al. (1980) Nucl. Acids Res. 12, 8711).
  • Such vectors and microorganisms transformed with them, in particular Agrobacterium are further objects of the present invention.
  • Agrobacteria transformed with a vector according to the invention can then be used in a known manner to transform plants, in particular crop plants, such as cereals, corn, soybeans, rice, cotton, sugar beet, canola, sunflower, flax, potato, tobacco, tomato, rape, alfalfa, lettuce and the various tree, nut and white species, for example by wounding Leaves or pieces of leaf are bathed in an agrobacterial solution and then cultivated in suitable media.
  • crop plants such as cereals, corn, soybeans, rice, cotton, sugar beet, canola, sunflower, flax, potato, tobacco, tomato, rape, alfalfa, lettuce and the various tree, nut and white species, for example by wounding Leaves or pieces of leaf are bathed in an agrobacterial solution and then cultivated in suitable media.
  • transgenic plants are a further subject of the present invention.
  • the transformation of plants by agrobacteria is known, among other things, from F.F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S-d Kung and R. Wu, Academic Press, 1993, pp. 15-38 and from S.B. Gelvin, Molecular Genetics of T-DNA Transfer from Agrobacterium to Plants, also in Transgenic Plants, pp. 49-78. From the transformed cells of the wounded leaves or leaf pieces, transgenic plants can be regenerated in a known manner, which under the control of the introduced promoter express the gene fused with it in a leaf-specific manner. Such transgenic plants, propagation material thereof and plant cells, tissue or parts are a further subject of the present invention.
  • cloning steps carried out in the context of the present invention such as e.g. Restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking of DNA fragments, transformation of E. coli cells, cultivation of bacteria, multiplication of phages and sequence analysis of recombinant DNA were like in Sambrook et al. (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6).
  • the bacterial strains used below (E. coli, XL-1 Blue and P2 392) were purchased from Stratagene.
  • a reverse transcriptase-generated cDNA probe of the cy-FBPase gene from potatoes was used for the experiments described below.
  • the cDNA probe ( Figure 10) comprised 1487 nucleotides.
  • the region coding for the structural gene (FBPase) comprises nucleotides 199 to 1218.
  • the DNA was ligated into BamHI digested EMBL3 arms obtained from Stratagene (11099 North Torrey Pines Road, La Jolla, CA 92037, USA) according to the manufacturer's instructions and then packaged in vitro (Gigapack II Gold packaging extracts, Stratagene, according to the manufacturer's instructions).
  • E. coli bacteria of the P2 392 strain (Stratagene) were infected with the recombinant lambda phages, the titer of the bank determined and then the bank amplified.
  • the sample of the cy-FBPase from potato radiolabeled with High-Prime (Boehringer Mannheim) was added to the prehybridization solution after 5 minutes of denaturation at 95 ° C.
  • the filters were hybridized overnight at 42 ° C. After the radioactive hybridization solution had been stripped off, the filters were washed for 20 min at 42 ° C. in 2X SSC (a NaCl / NaCitrate buffer), 0.1% SDS. It was then washed again with IX SSC, 0.1% SDS for 20 min at the same temperature. A film was then placed on the filters and exposed overnight at -70 ° C.
  • phage DNAs were visualized by autoradiography and isolated. Starting from a single plaque each, a lytic agar plate was inoculated, incubated overnight at 37 ° C. and the phages washed away the next day with 10 ml phage buffer (SM). The phage supernatant was then mixed with chloroform and the bacteria were centrifuged off. A spatula tip DNase and RNase were added to the supernatant and incubated at 37 ° C for 30 min. After adding 100 ⁇ l 0.5 M EDTA and 200 ⁇ l 10% SDS solution, the mixture was incubated for a further 20 minutes at 65 ° C.
  • SM ml phage buffer
  • the fragment lengths of the promoter regions of the 4 isolated clones were determined by Southern hybridizations with a 5 'cDNA sample after different restriction cleavages. Clone FBP-1 was selected for further analysis. An approximately 7100 b BamHI fragment of the clone FBP-1 was cloned into the BamHI site of the vector pUC19 for further characterization. The promoter region could be restricted to a 1724 base pair fragment by sequencing, Southern hybridization and restriction analysis (FIG. 1A). The 5 '-342 bp (Hindi / EcoRI) and 3' -216 bp (EcoRI / EcoRV) subfragments of the cDNA of the cy-FBPase served as a probe for the Southern hybridization.
  • the expression properties of the new promoter were analyzed by marker gene experiments.
  • the cy-FBPase promoter was fused with the ß-glucuronidase gene (GUS) from E. coli.
  • the promoter was isolated as a BamHI fragment from the plasmid FBP: pBlue and cloned into the BamHI site of the expression vector pBIlOl (FIG. 3A) (Jefferson et al.,
  • a deletion construct was made up comprising approximately 1.1 kb of the promoter sequence.
  • a fragment was extracted from FBP: GUS using EcoRI digestion, which comprises the GUS gene, the NOS terminator and 1100 bp of the promoter. This fragment was isolated, purified and ligated into the EcoRI site of the vector pBin 19 ( Figure 3A). The orientation of the fragment in pBin 19 was checked by cleavage with BamHI. Agrobacterium tumefaciens was also transformed with this vector.
  • Leaf disks of sterile plants were bathed in this bacterial solution in a sterile petri dish. The leaf disks were then placed in Petri dishes on MS medium (Murashige and Skoog, Physiol. Plant.
  • 60 transformed plants were regenerated from each of the transformed tobacco and potato plants and the ⁇ -glucuronidase activity was determined.
  • the ⁇ -glucuronidase detection was carried out as 45 by Martin et al. (1992) in: The GUS Reporter System as a Tool to Study Plant Gene Expression in: GUS Protoc-ols: Using the GUS Gene as a Reporter of Gene Expression, Academic Press, pp. 23-43 wrote. 40 tobacco plants and 21 potato plants were selected for a more detailed analysis of the expression. After transfer of the transformed plants into the greenhouse, the organ-specific expression of the ⁇ -glucuronidase was determined.
  • FIG. 4 shows a comparison of the enzyme activities in different potato tissues. It can be seen from the data that the promoter mediates leaf-specific expression of the reporter gene.
  • FIG. 5 compares the GUS activity in various wild-type organs and transgenic tobacco plants which carry the GUS gene under the control of various promoters according to the invention.
  • TME-1/67 denotes a plant transformed with FBP: GUS.
  • TME-11/13 denotes a plant transformed with FBP: GUS (DEL).
  • the determination of the GUS activity in the organs sink and source leaf, stem and root of 9-week-old tobacco plants and seeds showed that in both transformation lines the highest activity can be found in source leaves and a significantly lower one in sink leaves .
  • the measured values in sink leaves were always very different within one leaf.
  • the activity in stem and root was only slightly above the background activity measured in wild type tobacco.
  • the promoter is not active in these tissues. Slightly increased activities could be measured in tobacco seeds compared to wild-type seeds, although no mRNA was detectable in the "Northern blot”.
  • GUS activity in seeds was also found when incubating seed homogenate in X-Gluc solution. Wild-type seeds showed no coloration here (not shown).
  • the promoter may be active in the course of seed development and not in the mature seeds themselves. GUS activity could be due to stored protein. Depending on the plant, the activity of the leaves was increased by a factor of about 10 to 50 compared to the seed.
  • FIG. 6 illustrates the cell specificity of the FBP: GUS construct according to the invention. Identical histological findings are obtained with the shortened promoter construct
  • FBP FBP: CIS (DEL).
  • CIS DEL
  • leaf cross sections of fully unfolded tobacco leaves were made in the greenhouse of 8-week-old plants. The sections were fixed in 3% paraformaldehyde for 20 min, stained overnight in X-gluc (5-bromo-4-chloro-3-indolyl-ß-D-glucuronide) solution and then the chlorophyll with 7% ethanol removed.
  • X-gluc 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide
  • the chlorophyll with 7% ethanol removed.
  • the epider withdrawn from mesophyll and incubated separately to avoid contamination by dibromo-dichloro-indigo, which had been released into the staining solution by damaged mesophyll cells.
  • FIG. 6 (A) shows a cross section through the central conductive tissue of a source sheet. It can be clearly seen that only the mesophyll cells on the top were stained in the central lead tissue. The parenchymatic tissue, as well as xylem, phloem and other tissues localized here were not stained. Some of the epidermal cells appeared to be stained. This was probably not activity in the cells, because the isolated, incubated epidermis showed no GUS activity in trichomes, stomata and epidermal cells (B). The staining of the epidermis cells in (A) could be due to contamination from cut-out mesophyll cells or because the sections were multilayered and mesophyll cells behind the epidermis cells shone through. There was no blue color in the petiole (C). The cross-section through the mesophyll showed very strong expression in the palisade parenchyma and somewhat less in the sponge parenchyma (D).
  • the promoter fragment was cut out of pUC 19 with BamHI / SacI; the ends were filled in with T4 DNA polymerase. The fragment was then electrophoresed from the vector pUC19 using a 1% TAE agarose gel and purified with glass milk (BIO 101.1070 Joshua Way, Vista, CA 92083, USA). It was then ligated into an EcoRI cut pBin19 derivative treated with Klenow enzyme. This derivative thus contained an expression cassette comprising the cy-FBPase promoter and the terminator sequence of the octopine synthase from Agrobacterium tumefaciens (FIG. 9).
  • MOLECULE TYPE Genomic DNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • MOIE COOL TYPE Genomic DNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • CTGTTGAGTA CCATTGATCC GTC ⁇ ATCTTG TCGATAACTT TGATAAGGAT ATTTCAGGCA 240
  • ATCCCATGCA TCACTCTGCT CTTTCCACGT GGCATCCTCT GACGTCAGAT CAGATTCCTC 1680
  • MOLECULE TYPE Genomic DNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • AAACAATTTA TTTATTACTC CTATCCAATT CATTATATTT TCAAAAGTTA TGAAGTCCAC 420
  • MOLECULE TYPE Genomic DNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • MOLECULE TYPE Genomic DNA
  • HYPOTHETICAL NO
  • ANTISENSE NO

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Abstract

L'invention concerne des promoteurs qui provoquent l'expression permanente et spécifique aux feuilles d'une séquence de nucléotides de codage contrôlée par ces promoteurs, par exemple d'une séquence qui confère une résistance ou augmente la capacité de photosynthèse.
PCT/EP1997/005900 1996-10-25 1997-10-24 Expression de genes specifiques aux feuilles dans des plantes transgeniques WO1998018940A1 (fr)

Priority Applications (8)

Application Number Priority Date Filing Date Title
AU69091/98A AU743558B2 (en) 1996-10-25 1997-10-24 Leaf-specific gene expression in transgenetic plants
EP97948804A EP0938569B1 (fr) 1996-10-25 1997-10-24 Expression de genes specifiques aux feuilles dans des plantes transgeniques
US09/284,418 US6229067B1 (en) 1996-10-25 1997-10-24 Leaf-specific gene expression in transgenetic plants
JP10520030A JP2001502551A (ja) 1996-10-25 1997-10-24 トランスジェニック植物における遺伝子の葉部特異的発現
BR9712656-0A BR9712656A (pt) 1996-10-25 1997-10-24 Promotor, uso do mesmo, cassete de expressão, vetor recombinante, uso do mesmo, microorganismo, uso do mesmo, planta transgênica, processo de produção da mesma, processo de isolamento de um promotor especìfico para folha, e, sequência de ácido nucléico.
CA2268860A CA2268860C (fr) 1996-10-25 1997-10-24 Expression de genes specifiques aux feuilles dans des plantes transgeniques
DE59712978T DE59712978D1 (de) 1996-10-25 1997-10-24 Blattspezifische expression von genen in transgenen pflanzen
IL12946497A IL129464A (en) 1996-10-25 1997-10-24 Leaf-specific gene expression in transgenic plants

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DE19644478.0 1996-10-25
DE19644478A DE19644478A1 (de) 1996-10-25 1996-10-25 Blattspezifische Expression von Genen in transgenen Pflanzen

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US09/799,895 Division US6610840B2 (en) 1996-10-25 2001-03-07 Method of isolating a mesophyll-specific promoter

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DE (2) DE19644478A1 (fr)
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WO2005054453A1 (fr) 2003-12-02 2005-06-16 Basf Aktiengesellschaft 2-methyl-6-solanylbenzoquinone methyltransferase utilisee comme cible pour des herbicides
WO2005085454A3 (fr) * 2004-03-03 2005-11-24 Nat Univ Corp Nara Inst Procédé pour améliorer la productivité de plantes par technologie chloroplaste
WO2006111512A1 (fr) 2005-04-19 2006-10-26 Basf Plant Science Gmbh Methodes ameliorees controlant une expression genique
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DE102008014041A1 (de) 2008-03-13 2009-09-17 Leibniz-Institut für Pflanzengenetik Und Kulturpflanzenforschung (IPK) Verfahren zur Erzeugung einer Breitband-Resistenz gegenüber Pilzen in transgenen Pflanzen
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IL129464A (en) 2005-09-25
DE19644478A1 (de) 1998-04-30
CA2268860A1 (fr) 1998-05-07
DE59712978D1 (de) 2008-12-11
CA2268860C (fr) 2010-05-11
EP0938569A1 (fr) 1999-09-01
AU6909198A (en) 1998-05-22
BR9712656A (pt) 1999-10-26
EP0938569B1 (fr) 2008-10-29
US6610840B2 (en) 2003-08-26
US6229067B1 (en) 2001-05-08
AU743558B2 (en) 2002-01-31
US20020120955A1 (en) 2002-08-29
ATE412752T1 (de) 2008-11-15
JP2001502551A (ja) 2001-02-27
IL129464A0 (en) 2000-02-29

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